Mouse Hepatitis Virus Research Articles

Effects of immunoproteasome inhibition on acute respiratory infection with murine hepatitis virus strain 1.

Inflammatory responses triggered by acute infection by respiratory viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) drive morbidity and mortality. Infection of mice with murine hepatitis virus strain 1 (MHV-1), a mouse coronavirus, is a useful model to study the pathogenesis of coronavirus respiratory infections. The immunoproteasome is an inducible component of the ubiquitin proteasome system that is poised to contribute to multiple aspects of immune function, inflammation, and protein homeostasis during an infection. We used the MHV-1 model to define the role of the immunoproteasome in coronavirus pathogenesis. We found that immunoproteasome subunit expression increases in the lungs and the liver during acute MHV-1 respiratory infection. Inhibition of immunoproteasome activity did not affect MHV-1 replication but increased MHV-1-induced weight loss, mortality, and inflammation in lungs and livers. Thus, our findings indicate that the immunoproteasome is a critical protective host factor during coronavirus respiratory infection.

Biochemical analysis of packing and assembling heptad repeat motifs in the coronavirus spike protein trimer.

During a coronavirus infection, the spike protein undergoes sequential structural transitions triggered by its receptor and the host protease at the interface between the virus and cell membranes, thereby mediating membrane fusion. After receptor binding, the heptad repeat motif (HR1/HR2) within the viral spike protein bridges the viral and cellular membranes; however, the intermediate conformation adopted by the spike protein when drawing the viral and cellular membranes into close proximity remains unclear due to its transient and unstable nature. Here, we experimentally induced conformational changes in the spike protein of a murine coronavirus by incubating the virus with its receptor, followed by exposure to trypsin. We then treated the virus/receptor complex with proteinase K to probe the tightly packed core structure of the spike protein. The conformations of the spike protein were predicted from the sizes of the protease digestion products detected by western blot analysis. Upon receptor binding, two bands (each showing different reactivity with a fusion-inhibiting HR2-peptide) were detected; we propose that these bands correspond to the packed and unpacked HR1/HR2 motifs. After trypsin-mediated triggering, measurement of temperature and time dependency revealed that packing of the remaining unpacked HR1/HR2 motifs and assembly of three HR1 motifs in a trimer occur almost simultaneously. Thus, the trimeric spike protein adopts an asymmetric-unassembled conformation after receptor binding, followed by direct assembly into the post-fusion form triggered by the host protease. This biochemical study provides mechanistic insight into the previously unknown intermediate structure of the viral fusion protein.IMPORTANCEDuring infection by an enveloped virus, receptor binding triggers fusion between the cellular membrane and the virus envelope, enabling delivery of the viral genome to the cytoplasm. The viral spike protein mediates membrane fusion; however the molecular mechanism underlying this process is unclear. This is because using structural biology methods to track the transient conformational changes induced in the unstable spike trimer is challenging. Here, we harnessed the ability of protease enzymes to recognize subtle differences on protein surfaces, allowing us to detect structural differences in the spike protein before and after conformational changes. Differences in the size of the degradation products were analyzed by western blot analysis. The proposed model explaining the conformational changes presented herein is a plausible candidate that provides valuable insight into unanswered questions in the field of virology.

Hidden evolutionary constraints dictate the retention of coronavirus accessory genes.

Coronaviruses exhibit many mechanisms of genetic innovation, including the acquisition of accessory genes that originate by capture of cellular genes or through duplication of existing viral genes. Accessory genes influence viral host range and cellular tropism, but little is known about how selection acts on these variable regions of virus genomes. We used experimental evolution of mouse hepatitis virus (MHV) encoding a cellular AKAP7 phosphodiesterase and an inactive native phosphodiesterase, NS2 to model the evolutionary fate of accessory genes. After courses of serial infection, the gene encoding inactive NS2, ORF2, unexpectedly remained intact, suggesting it is under cryptic constraint uncoupled from the function of NS2. In contrast, AKAP7 was retained under strong selection but rapidly lost under relaxed selection. Experimental evolution also led to altered viral replication in a cell type-specific manner and changed the relative proportions of subgenomic viral RNA in plaque-purified viral isolates, revealing additional mechanisms of adaptation. Guided by the retention of ORF2 and similar patterns in related betacoronaviruses, we analyzed ORF8 of SARS-CoV-2, which arose via gene duplication and contains premature stop codons in several globally successful lineages. As with MHV ORF2, the coding-defective SARS-CoV-2 ORF8 gene remains largely intact, mirroring patterns observed during MHV experimental evolution, challenging assumptions on the dynamics of gene loss in virus genomes and extending these findings to viruses currently adapting to humans.

Coronavirus envelope protein activates TMED10-mediated unconventional secretion of inflammatory factors

The precise cellular mechanisms underlying heightened proinflammatory cytokine production during coronavirus infection remain incompletely understood. Here we identify the envelope (E) protein in severe coronaviruses (SARS-CoV-2, SARS, or MERS) as a potent inducer of interleukin-1 release, intensifying lung inflammation through the activation of TMED10-mediated unconventional protein secretion (UcPS). In contrast, the E protein of mild coronaviruses (229E, HKU1, or OC43) demonstrates a less pronounced effect. The E protein of severe coronaviruses contains an SS/DS motif, which is not present in milder strains and facilitates interaction with TMED10. This interaction enhances TMED10-oligomerization, facilitating UcPS cargo translocation into the ER-Golgi intermediate compartment (ERGIC)—a pivotal step in interleukin-1 UcPS. Progesterone analogues were identified as compounds inhibiting E-enhanced release of proinflammatory factors and lung inflammation in a Mouse Hepatitis Virus (MHV) infection model. These findings elucidate a molecular mechanism driving coronavirus-induced hyperinflammation, proposing the E-TMED10 interaction as a potential therapeutic target to counteract the adverse effects of coronavirus-induced inflammation.

The coronavirus nsp14 exoribonuclease interface with the cofactor nsp10 is essential for efficient virus replication and enzymatic activity.

Coronavirus replication requires assembly of a replication transcription complex composed of nonstructural proteins (nsp), including polymerase, helicase, exonuclease, capping enzymes, and non-enzymatic cofactors. The coronavirus nsp14 exoribonuclease mediates several functions in the viral life cycle including genomic and subgenomic RNA synthesis, RNA recombination, RNA proofreading and high-fidelity replication, and native resistance to many nucleoside analogs. The nsp-14 exonuclease activity in vitro requires the non-enzymatic co-factor nsp10, but the determinants and importance the nsp14-10 interactions during viral replication have not been defined. Here we show that for the coronavirus murine hepatitis virus, nsp14 residues at the nsp14-10 interface are essential for efficient viral replication and in vitro exonuclease activity. These results shed new light on the requirements for protein interactions within the coronavirus replication transcription complex, and they may reveal novel non active-site targets for virus inhibition and attenuation.

Neurotropic Murine β-Coronavirus Infection Causes Differential Expression of Connexin 47 in Oligodendrocyte Subpopulations Associated with Demyelination.

Gap junctions (GJs) play a crucial role in the survival of oligodendrocytes and myelination of the central nervous system (CNS). In this study, we investigated the spatiotemporal changes in the expression of oligodendroglial GJ protein connexin 47 (Cx47), its primary astroglial coupling partner, Cx43, and their association with demyelination following intracerebral infection with mouse hepatitis virus (MHV). Neurotropic strains of MHV, a β-coronavirus, induce an acute encephalomyelitis followed by a chronic demyelinating disease that shares similarities with the human disease multiple sclerosis (MS). Our results reveal that Cx47 GJs are persistently lost in mature oligodendrocytes, not only in demyelinating lesions but also in surrounding normal appearing white and gray matter areas, following an initial loss of astroglial Cx43 GJs during acute infection. At later stages after viral clearance, astroglial Cx43 GJs re-emerge but mature oligodendrocytes fail to fully re-establish GJs with astrocytes due to lack of Cx47 GJ expression. In contrast, at this later demyelinating stage, the increased oligodendrocyte precursor cells appear to exhibit Cx47 GJs. Our findings further highlight varying degrees of demyelination in distinct spinal cord regions, with the thoracic cord showing the most pronounced demyelination. The regional difference in demyelination correlates well with dynamic changes in the proportion of different oligodendrocyte lineage cells exhibiting differential Cx47 GJ expression, suggesting an important mechanism of progressive demyelination even after viral clearance.

Antiviral effect of oversulfated kappa-carrageenan derivatives against COVID-19 for spray coating application on facemasks

The COVID-19 pandemic caused by SARS-CoV-2 has spurred the urgent need for effective antiviral strategies. In this work, we explored the potential of oversulfated kappa-carrageenan (OSKC) in spray-coated facemasks for SARS-CoV-2 inhibition pathway. The sulfated derivative was synthesized with sulfur trioxide pyridine complex in dimethylformamide solution. The antiviral efficacy of OSKC at different concentrations and spray-coated facemasks was evaluated using betacoronavirus Murine Hepatitis Virus strain 3, revealing a significant reduction in viral load compared to commercial kappa-carrageenan. Furthermore, the characterization techniques assessed the effect of the position of the introduced sulfate groups on the antiviral activity and on the physicochemical characteristics. OSKC is able to bind specific proteins of enveloped viruses, preventing viral attachment into target cells. Overall, this study demonstrates the feasibility and effectiveness of OSKC spray coating for breathable facemasks with antimicrobial properties, offering a promising approach to enhancing personal protective equipment against viral transmission in healthcare and community settings.

The Role of Coronavirus Spike Protein in Inducing Optic Neuritis in Mice: Parallels to the SARS-CoV-2 Virus.

Optic neuritis (ON), one of the clinical manifestations of the human neurological disease multiple sclerosis (MS), was also reported in patients with COVID-19 infection, highlighting one potential neurological manifestation of SARS-CoV-2. However, the mechanism of ON in these patients is poorly understood. Insight may be gained by studying the neurotropic mouse hepatitis virus (MHV-A59), a β-coronavirus that belongs to the same family as SARS-CoV-2. Mouse hepatitis virus-A59, or its isogenic spike protein recombinant strains, inoculation in mice provides an important experimental model to understand underpinning mechanisms of neuroinflammatory demyelination in association with acute stage optic nerve inflammation and chronic stage optic nerve demyelination concurrent with axonal loss. Spike is a surface protein that mediates viral binding and entry into host cells, as well as cell-cell fusion and viral spread. Studies have implicated spike-mediated mechanisms of virus-induced neuroinflammatory demyelination by comparing naturally occurring demyelinating (DM) and nondemyelinating (NDM) MHV strains. Here, we summarize findings in MHV-induced experimental ON and myelitis, using natural DM and NDM strains as well as engineered recombinant strains of MHV to understand the role of spike protein in inducing ON and demyelinating disease pathology. Potential parallels in human coronavirus-mediated ON and demyelination, and insight into potential therapeutic strategies, are discussed.

Performance of three multiplex real-time PCR assays for simultaneous detection of 12 infectious pathogens in mice affected with respiratory and digestive diseases.

Research quality can be improved with reliable and reproducible experimental results when animal experiments are conducted using laboratory animals with guaranteed microbiological and genetic quality through health monitoring. Therefore, health monitoring requires the rapid and accurate diagnosis of infectious diseases in laboratory animals. This study presents a performance evaluation of a commercially available multiplex real-time PCR (mRT-PCR) assay for the rapid detection of 12 infectious pathogens (Set 1: Sendai virus [SeV, formally murine respirovirus], Mycoplasma spp., Rodentibacter pneumotropicus, and Rodentibacter heylii; Set 2: Helicobacter spp., Murine norovirus [MNV], Murine hepatitis virus [MHV], and Salmonella spp.; Set 3: Staphylococcus aureus, Streptobacillus moniliformis, Corynebacterium kutscheri, and Pseudomonas aeruginosa). To evaluate the efficacy of the mRT-PCR assay, 102 clinical samples encompassing fecal and cecal specimens were analyzed. The resulting data were then compared with the findings from sequence analysis for validation. The assay's detection limit ranged from 1 to 100 copies per reaction. Specificity testing involving various viruses and bacteria indicated no cross-reactivity between strains. Additionally, the assay exhibited good reproducibility, with mean coefficients of variation for inter- and intra assay variation below 3%. The overall positive rate was 52.9% (n = 54), with the mRT-PCR assay findings matching sequence analysis results (κ = 1). MHV (n = 29, 28.4%) was the most prevalent pathogen, followed by Helicobacter spp. (n = 28, 27.5%), R. heylii (n = 18, 17.6%), Mycoplasma spp. (n = 14, 13.7%), MNV (n = 12, 11.8%), S. aureus (n = 9, 8.8%), P. aeruginosa (n = 4, 3.9%), and R. pneumotropicus (n = 1, 0.9%). This assay offers a rapid turnaround time of 100 min, including 30 min for DNA preparation and 70 min for target DNA/RNA amplification. It ensures accuracy, minimizing false positives or negatives, making it a convenient tool for the simultaneous detection of infectious diseases in many samples. Overall, the propose‑d assay holds promise for the effective detection of the most important pathogens in laboratory animal health monitoring.

Mouse hepatitis virus JHMV I protein is required for maximal virulence.

Betacoronaviruses encode a conserved accessory gene within the +1 open reading frame (ORF) of nucleocapsid called the internal N gene. This gene is referred to as "I" for mouse hepatitis virus (MHV), ORF9b for severe acute respiratory CoV (SARS-CoV) and SARS-CoV-2, and ORF8b for Middle East respiratory syndrome CoV (MERS-CoV). Previous studies have shown ORF8b and ORF9b have immunoevasive properties, while the only known information for MHV I is its localization within the virion of the hepatotropic/neurotropic A59 strain of MHV. Whether MHV I is an innate immune antagonist or has other functions has not been evaluated. In this report, we show that the I protein of the neurotropic JHM strain of MHV (JHMV) lacks a N terminal domain present in other MHV strains, has immunoevasive properties, and is a component of the virion. Genetic deletion of JHMV I (rJHMVIΔ57-137) resulted in a highly attenuated virus both in vitro and in vivo that displayed a post RNA replication/transcription defect that ultimately resulted in fewer infectious virions packaged compared with wild-type virus. This phenotype was only seen for rJHMVIΔ57-137, suggesting the structural changes predicted for A59 I altered its function, as genetic deletion of A59 I did not change viral replication or pathogenicity. Together, these data show that JHMV I both acts as a mild innate immune antagonist and aids in viral assembly and infectious virus production, and suggest that the internal N proteins from different betacoronaviruses have both common and virus strain-specific properties.IMPORTANCECoV accessory genes are largely studied in overexpression assays and have been identified as innate immune antagonists. However, functions identified after overexpression are often not confirmed in the infected animal host. Furthermore, some accessory proteins are components of the CoV virion, but their role in viral replication and release remains unclear. Here, we utilized reverse genetics to abrogate expression of a conserved CoV accessory gene, the internal N ("I") gene, of the neurotropic JHMV strain of MHV and found that loss of the I gene resulted in a post replication defect that reduced virion assembly and ultimately infectious virus production, while also increasing some inflammatory molecule expression. Thus, the JHMV I protein has roles in virion assembly that were previously underappreciated and in immunoevasion.

HSP70 contributes to pathogenesis of fulminant hepatitis induced by coronavirus

Fulminant viral hepatitis (FH) represents a significant clinical challenge, with its pathogenesis not yet fully elucidated. Heat shock protein (HSP)70, a molecular chaperone protein with a broad range of cytoprotective functions, is upregulated in response to stress. However, the role of HSP70 in FH remains to be investigated. Notably, HSP70 expression is upregulated in the livers of coronavirus-infected mice and patients. Therefore, we investigated the mechanistic role of HSP70 in coronavirus-associated FH pathogenesis. FH was induced in HSP70-deficient (HSP70 KO) mice or in WT mice treated with the HSP70 inhibitor VER155008 when infected with the mouse hepatitis virus strain A59 (MHV-A59). MHV-A59-infected HSP70 KO mice exhibited significantly reduced liver damage and mortality. This effect was attributed to decreased infiltration of monocyte-macrophages and neutrophils in the liver of HSP70 KO mice, resulting in lower levels of inflammatory cytokines such as IL-1β, TNFα, and IL-6, and a reduced viral load. Moreover, treatment with the HSP70 inhibitor VER155008 protected mice from MHV-A59-induced liver damage and FH mortality. In summary, HSP70 promotes coronavirus-induced FH pathogenesis by enhancing the infiltration of monocyte-macrophages and neutrophils and promoting the secretion of inflammatory cytokines. Therefore, HSP70 is a potential therapeutic target in viral FH intervention.

Chromatin Regulator SMARCA4 Is Essential for MHV-Induced Inflammatory Cell Death, PANoptosis.

The innate immune system serves as the first line of defense against β-coronaviruses (β-CoVs), a family of viruses that includes SARS-CoV-2. Viral sensing via pattern recognition receptors triggers inflammation and cell death, which are essential components of the innate immune response that facilitate viral clearance. However, excessive activation of the innate immune system and inflammatory cell death can result in uncontrolled release of proinflammatory cytokines, resulting in cytokine storm and pathology. PANoptosis, innate immune, inflammatory cell death initiated by innate immune sensors and driven by caspases and RIPKs through PANoptosome complexes, has been implicated in the pathology of viral infections. Therefore, understanding the molecular mechanisms regulating PANoptosis in response to β-CoV infection is critical for identifying new therapeutic targets that can mitigate disease severity. In the current study, we analyzed findings from a cell death-based CRISPR screen with archetypal β-CoV mouse hepatitis virus (MHV) as the trigger to characterize host molecules required for inflammatory cell death. As a result, we identified SMARCA4, a chromatin regulator, as a putative host factor required for PANoptosis in response to MHV. Furthermore, we observed that gRNA-mediated deletion of Smarca4 inhibited MHV-induced PANoptotic cell death in macrophages. These findings have potential translational and clinical implications for the advancement of treatment strategies for β-CoVs and other infections.

CD6 Regulates CD4 T Follicular Helper Cell Differentiation and Humoral Immunity During Murine Coronavirus Infection.

During activation the T cell transmembrane receptor CD6 becomes incorporated into the T cell immunological synapse where it can exert both co-stimulatory and co-inhibitory functions. Given the ability of CD6 to carry out opposing functions, this study sought to determine how CD6 regulates early T cell activation in response to viral infection. Infection of CD6 deficient mice with a neurotropic murine coronavirus resulted in greater activation and expansion of CD4 T cells in the draining lymph nodes. Further analysis demonstrated that there was also preferential differentiation of CD4 T cells into T follicular helper cells, resulting in accelerated germinal center responses and emergence of high affinity virus specific antibodies. Given that CD6 conversely supports CD4 T cell activation in many autoimmune models, we probed potential mechanisms of CD6 mediated suppression of CD4 T cell activation during viral infection. Analysis of CD6 binding proteins revealed that infection induced upregulation of Ubash3a, a negative regulator of T cell receptor signaling, was hindered in CD6 deficient lymph nodes. Consistent with greater T cell activation and reduced UBASH3a activity, the T cell receptor signal strength was intensified in CD6 deficient CD4 T cells. These results reveal a novel immunoregulatory role for CD6 in limiting CD4 T cell activation and deterring CD4 T follicular helper cell differentiation, thereby attenuating antiviral humoral immunity.

Isolation and In Vitro Biological Evaluation of Triterpenes from Salacia grandifolia Leaves.

Salacia grandifolia is naturally found in the Atlantic Forest regions of Brazil. Despite the pharmacological potential of plants from the Salacia genus, phytochemical studies on this species have not been reported in literature. A new triterpene, 28-hydroxyfriedelane-3,15-dione (1), and seven known compounds (friedelan-3-one (2), friedelan-3β-ol (3), friedelane-3,15-dione (4), 15α-hydroxyfriedelan-3-one (5), 28-hydroxyfriedelan-3-one (6), 30-hydroxyfriedelan-3-one (7), and 29-hydroxyfriedelan-3-one (8)) were obtained from the hexane extract of Salacia grandifolia leaves. These isolated compounds and three extracts, hexane (EH), chloroform (EC), and ethyl acetate (EAE), were assessed for their potential biological activities, which consisted in the evaluation of antiviral activity against a murine coronavirus, mouse hepatitis virus 3 (MHV-3), antibacterial activity against the susceptible and methicillin-resistant Staphylococcus aureus (MRSA), and antileukemia activity against the THP-1 and K-562 cell lines. The extracts EH and EAE along with the triterpenes 1 and 6 exhibited moderate to high antiviral activity, with emphasis on 6, which presented an EC50 value of 2.9 ± 0.3 μM. None of the compounds presented antibacterial activity against the tested strains. The evaluated compounds 1, 4, 6 and 7 exhibited low cytotoxic activity against the tested leukemia cell lines. Taken together, this study comprises an overview for the potential of the Salacia grandifolia biological activities, including a new isolated triterpene.

The glycoprotein quality control factor Malectin promotes coronavirus replication and viral protein biogenesis.

Coronaviruses (CoV) rewire host protein homeostasis (proteostasis) networks through interactions between viral nonstructural proteins (nsps) and host factors to promote infection. With the emergence of SARS-CoV-2, it is imperative to characterize host interactors shared across nsp homologs. Using quantitative proteomics and functional genetic screening, we identify conserved proteostasis interactors of nsp2 and nsp4 that serve pro-viral roles during infection of murine hepatitis virus - a model betacoronavirus. We uncover a glycoprotein quality control factor, Malectin (MLEC), which significantly reduces infectious titers when knocked down. During infection, nsp2 interacts with MLEC-associated proteins and the MLEC-interactome is drastically altered, stabilizing association with the Oligosaccheryltransferase (OST) complex, a crucial component of viral glycoprotein production. MLEC promotes viral protein levels and genome replication through its quality control activity. Lastly, we show MLEC promotes SARS-CoV-2 replication. Our results reveal a role for MLEC in mediating CoV infection and identify a potential target for pan-CoV antivirals.

Mutation of a highly conserved isoleucine residue in loop 2 of several β-coronavirus macrodomains indicates that enhanced ADP-ribose binding is detrimental to infection.

All coronaviruses (CoVs) encode for a conserved macrodomain (Mac1) located in nonstructural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that binds and hydrolyzes mono-ADP-ribose from target proteins. Previous work has shown that Mac1 is important for virus replication and pathogenesis. Within Mac1, there are several regions that are highly conserved across CoVs, including the GIF (glycine-isoleucine-phenylalanine) motif. To determine how the biochemical activities of these residues impact CoV replication, the isoleucine and the phenylalanine residues were mutated to alanine (I-A/F-A) in both recombinant Mac1 proteins and recombinant CoVs, including murine hepatitis virus (MHV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The F-A mutant proteins had ADP-ribose binding and/or hydrolysis defects that led to attenuated replication and pathogenesis in cell culture and mice. In contrast, the I-A mutations had normal enzyme activity and enhanced ADP-ribose binding. Despite increased ADP-ribose binding, I-A mutant MERS-CoV and SARS-CoV-2 were highly attenuated in both cell culture and mice, indicating that this isoleucine residue acts as a gate that controls ADP-ribose binding for efficient virus replication. These results highlight the function of this highly conserved residue and provide unique insight into how macrodomains control ADP-ribose binding and hydrolysis to promote viral replication.

The transcriptome of MHV-infected RAW264.7 cells offers an alternative model for macrophage innate immunity research.

Macrophages are the primary innate immune cells encountered by the invading coronaviruses, and their abilities to initiate inflammatory reactions, to maintain the immunity homeostasis by differential polarization, to train the innate immune system by epigenic modification have been reported in laboratory animal research. In the current in vitro research, murine macrophage RAW 264.7 cell were infected by mouse hepatitis virus, a coronavirus existed in mouse. At 3-, 6-, 12-, 24-, and 48-h post infection (hpi.), the attached cells were washed with PBS and harvested in Trizol reagent. Then The harvest is subjected to transcriptome sequencing. The transcriptome analysis showed the immediate (3 hpi.) up regulation of DEGs related to inflammation, like Il1b and Il6. DEGs related to M2 differential polarization, like Irf4 showed up regulation at 24 hpi., the late term after viral infection. In addition, DEGs related to metabolism and histone modification, like Ezh2 were detected, which might correlate with the trained immunity of macrophages. The current in vitro viral infection study showed the key innated immunity character of macrophages, which suggested the replacement value of viral infection cells model, to reduce the animal usage in preclinical research.

Development of two novel on-site detection visualization methods for murine hepatitis virus based on the multienzyme isothermal rapid amplification

Murine hepatitis virus (MHV) infection is one of the most prevalent types of mice infection in laboratory. MHV could cause death in mice and even interfere with the results in animal experiments. Herein, we developed two isothermal approaches based on the Multienzyme Isothermal Rapid Amplification (MIRA), for rapid detection of MHV in conserved M gene. We designed and screened several pairs of primers and probes and the isothermal fluorescence detector was applied for the exonuclease Ⅲ reverse transcription MIRA (exo-RT-MIRA) assay. To further simplify the workflow, the portable fluorescence visualization instrument, also as a palm-sized handheld system, was used for the naked-eye exo-RT-MIRA assay. The amplification temperature and time were optimized. The assay could be processed well at 42 °C 20 min for the exo-RT-MIRA and the naked-eye exo-RT-MIRA assay. The limit of detection (LoD) of the exo-RT-MIRA assay was 43.4 copies/μL. The LoD of the naked-eye exo-RT-MIRA assay was 68.2 copies/μL. No nonspecific amplifications were observed in the two assays. A total of 107 specimens were examined by qPCR and two assays developed. The experimental results statistical analysis demonstrated that the exo-RT-MIRA assay with the qPCR yielded sufficient agreement with a kappa value of 1.000 (p < 0.0001). The results also exhibited a good agreement (kappa value, 0.961) (p < 0.0001) between the naked-eye exo-RT-MIRA assay and the qPCR assay. In our study, the exo-RT-MIRA assay and the naked-eye exo-RT-MIRA assay presented the possibility of new methods in MHV point-of-testing diagnosis.

Evaluation of RNA Secondary Stem-Loop Structures in the UTRs of Mouse Hepatitis Virus as New Therapeutic Targets.

MHV-A59 is a beta-coronavirus that causes demyelinating encephalitis and hepatitis in mice. Recently, the mouse infection model of MHV-A59 has been used as an alternative animal infection model for SARS-CoV and SARS-CoV-2, aiding the development of new antiviral drugs. In this study, the MHV-A59 model was employed to investigate the potential of SARS-CoV-2 UTRs as new targets for antiviral drugs. Optimal targets within the MHV-A59 UTRs were identified using a shRNA and siRNA design tool, focusing on RNA secondary stem-loop (SL) structures in the UTRs. We then examined whether the designed RNAi constructs could inhibit MHV-A59 replication. In the 5'UTR, the stem-loop 1 (SL1) was identified as the most effective target, while in the 3'UTR, the minimal element for the initiation of negative-strand RNA synthesis (MIN) proved to be the most effective. Importantly, siRNAs targeting SL1 and MIN structures significantly reduced total RNA synthesis, negative-strand genomic RNA synthesis, subgenomic (sg) RNA synthesis, viral titer, and the plaque size of MHV-A59 compared to the control. Although not statistically significant, the combination of siSL1 and siMIN had a stronger effect on inhibiting MHV-A59 replication than either siRNA monotherapy. Interestingly, while the SL1 structure is present in both MHV and SARS-CoV-2, the MIN structure is unique to MHV. Thus, the SL1 of SARS-CoV-2 may represent a novel and promising target for RNAi-based antiviral drugs.

Cystatin F attenuates neuroinflammation and demyelination following murine coronavirus infection of the central nervous system

BackgroundCystatin F is a secreted lysosomal cysteine protease inhibitor that has been implicated in affecting the severity of demyelination and enhancing remyelination in pre-clinical models of immune-mediated demyelination. How cystatin F impacts neurologic disease severity following viral infection of the central nervous system (CNS) has not been well characterized and was the focus of this study. We used cystatin F null-mutant mice (Cst7-/-) with a well-established model of murine coronavirus-induced neurologic disease to evaluate the contributions of cystatin F in host defense, demyelination and remyelination.MethodsWildtype controls and Cst7-/- mice were intracranially (i.c.) infected with a sublethal dose of the neurotropic JHM strain of mouse hepatitis virus (JHMV), with disease progression and survival monitored daily. Viral plaque assays and qPCR were used to assess viral levels in CNS. Immune cell infiltration into the CNS and immune cell activation were determined by flow cytometry and 10X genomics chromium 3’ single cell RNA sequencing (scRNA-seq). Spinal cord demyelination was determined by luxol fast blue (LFB) and Hematoxylin/Eosin (H&E) staining and axonal damage assessed by immunohistochemical staining for SMI-32. Remyelination was evaluated by electron microscopy (EM) and calculation of g-ratios.ResultsJHMV-infected Cst7-/- mice were able to control viral replication within the CNS, indicating that cystatin F is not essential for an effective Th1 anti-viral immune response. Infiltration of T cells into the spinal cords of JHMV-infected Cst7-/- mice was increased compared to infected controls, and this correlated with increased axonal damage and demyelination associated with impaired remyelination. Single-cell RNA-seq of CD45 + cells enriched from spinal cords of infected Cst7-/- and control mice revealed enhanced expression of transcripts encoding T cell chemoattractants, Cxcl9 and Cxcl10, combined with elevated expression of interferon-g (Ifng) and perforin (Prf1) transcripts in CD8 + T cells from Cst7-/- mice compared to controls.ConclusionsCystatin F is not required for immune-mediated control of JHMV replication within the CNS. However, JHMV-infected Cst7-/- mice exhibited more severe clinical disease associated with increased demyelination and impaired remyelination. The increase in disease severity was associated with elevated expression of T cell chemoattractant chemokines, concurrent with increased neuroinflammation. These findings support the idea that cystatin F influences expression of proinflammatory gene expression impacting neuroinflammation, T cell activation and/or glia cell responses ultimately impacting neuroinflammation and neurologic disease.